The invention relates to expression of trehalose biosynthetic genes and drought resistance in plants.
Trehalose (a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside) is a disaccharide commonly found in lower organisms such as bacteria, fungi and insects where it often accumulates in resting or stationary phase cells and organs. Two enzymatic activities are required for trehalose biosynthesis: a trehalose-6-phosphate synthase catalyses the condensation of UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate and a trehalose-6-phosphate phosphatase phosphorylates trehalose-6-phosphate to trehalose.
Although trehalose can serve as a storage form for reduced carbon, it may play a more significant role as a protectant against the deleterious effects of various abiotic stresses, notably heat and desiccation. Both in vivo and in vitro, trehalose accumulation is correlated with protection of biological macromolecules (particularly membranes and proteins) from dessication, temperature extremes, and osmotic shock. Trehalose produced by fermentation is used commercially in the preservation of enzymes and is registered as a food additive for the stabilization of dehydrated and processed foods.
While it has long been recognized that trehalose may occur in plants as a product of symbiotic microorganisms, as a rule vertebrates and higher plants were thought not to be capable of synthesizing trehalose. The near-ubiquitous occurrence of specific trehalose-catabolizing enzymes (trehalases) in higher plant families was a biological curiousity ascribed mainly to the presence of exogenous trehalose entering plant cells from symbiotic or epiphytic microbial and fungal sources. Notable exceptions are the lower plants and angiosperms grouped loosely into the category of xe2x80x9cresurrection plantsxe2x80x9d which are capable of surviving extraordinarily prolonged periods of dessication. These plants, including species of Selaginella and Myrothamnus, can accumulate as much as 10% trehalose by dry weight following the onset of droughting.
In view of trehalose""s association with drought resistance and the historically poor economics of microbial trehalose fermentation, attempts have also been made to engineer transgenic plants to accumulate this disaccharide. Although such plants have been obtained, it has become apparent that constitutive trehalose production in the plant cytosol is accompanied by significant deleterious effects. These phenotypes (stunted growth, abnormal leaves, undeveloped roots) are particulary severe when trehalose expression occurs in root tissue or during early development, as the use of green-tissue specific plant promoters to drive trehalose producing genes ameliorates some, but not all, of these effects.
Given these facts, an inducible expression system for the trehalose biosynthetic genes, which allows for trehalose accumulation and results in drought resistance but without deleterious effects to the plant is of great practical use and economic interest.
The present invention thus relates to expression of trehalose biosynthetic genes and drought resistance in plants. In particular, this invention addresses the issue of trehalose accumulation and drought resistance in higher plants and novel ways to engineer such trait. It also addresses the need for improved storage properties of harvested plants, improved shelf-life of fruits and flowers, as well as stabilization of foreign proteins expressed in transgenic plants. In a prefered embodiment, the invention describes the expression of the trehalose biosynthetic genes in plants, preferably under the control of an inducible promoter, which allow for drought resistance without the deleterious effect associated with uncontrolled accumulation of trehalose. A prefered promoter is a chemically inducible promoter, such as the tobacco PR-1a promoter, which can be activated by foliar application of a chemical inducer.
Additionally, the invention describes expression of the trehalose biosynthetic genes expressed in different cellular compartments. In a first embodiment the trehalose biosynthetic genes are expressed in the plant cytoplasm. In a further embodiment, the trehalose biosynthetic genes are expressed from the plant nuclear genome and the trehalose biosynthetic enzymes encoded therefrom are targeted to the plastids, e.g. by using a plastid transit peptide. In a further embodiment, the trehalose biosynthetic genes are expressed from the plant plastid genome. In a preferred embodiment, vectors containing the trehalose biosynthetic genes fused to a promoter capable of directing the expression of the trehalose biosynthetic genes in plant plastids are transformed into the plastid genome. In a preferred embodiment, vectors containing a phage promoter fused to the trehalose biosynthetic genes are transformed into the plastid genome. The resulting line is crossed to a transgenic line containing a nuclear coding region for a phage RNA polymerase supplemented with a plastid-targeting sequence and operably linked to a plant promoter, such as an inducible promoter, a tissue-specific promoter or a constitutive promoter. In another preferred embodiment, a promoter capable of directing the expression of the trehalose biosynthetic genes in plant plastids is a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase. Such promoters are e.g. but not limited to a clpP promoter, a 16S r-RNA gene promoter, a psbA promoter or a rbcL promoter.
In the present invention, trehalose biosynthetic genes from E. coli are preferably used, but trehalose biosynthetic genes from other organisms including but not limited to yeast, other lower organisms or higher organisms, such as plants, are suitable. In a preferred embodiment, a nucleotide sequence encoding a trehalose phosphate synthase and a nucleotide sequence encoding a trehalose phosphate phosphatase are both expressed in the plant. In another preferred embodiment, a nucleotide sequence encoding a trehalose phosphate synthase is expressed in the plant, or a nucleotide sequence encoding a trehalose phosphate phosphatase is expressed in the plant. The present invention also relates to the expression from the plastid genome of two trehalose biosynthetic genes transcribed from a single promoter in an operon-like polycistronic gene.
The invention thus provides:
A plant expressing a nucleotide sequence encoding a trehalose biosynthetic enzyme, for example a plant comprising nucleotide sequence coding for the trehalose 6-phosphate synthase and/or trehalose 6-phosphate phosphatase, for example the E. coli OtsA and/or E. coli OtsB genes. Such nucleotide sequences are for example stably integrated in its nuclear or plastid genome, under the control of a promoter capable of directing the expression of the trehalose biosynthetic genes in said plant, e.g. under the control of an inducible promoter, e.g., a wound-inducible or chemically inducible promoter, such as the tobacco PR-1a promoter or Arabidopsis PR-1 promoter, or, a transactivator-regulated promoter wherein the corresponding transactivator is under the control of a promoter capable of directing the expression of the transactivator in said plant, e.g. an inducible promoter, a tissue-specific promoter or a constitutive promoter, e.g., a wound-inducible or chemically inducible promoter, such as the tobacco PR-1a promoter or Arabidopsis PR-1 promoter;
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag with instructions for use. In particular, the invention provides:
A plant comprising in its nuclear genome a first heterologous expression cassette or parts thereof comprising a nucleotide sequence encoding a trehalose 6-phosphate synthase under control of an inducible promoter and a second heterologous expression cassette or parts thereof comprising a nucleotide sequence encoding a trehalose-6-phosphate phosphatase under control of an inducible promoter. also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag with instructions for use.
The invention further provides:
A plant comprising in its plastid genome a first heterologous expression cassette or parts thereof comprising a nucleotide sequence encoding a trehalose 6-phosphate synthase under control of a promoter capable of directing the expression of the nucleotide sequence in the plastids of said plant, e.g. a transactivator-regulated promoter wherein the corresponding transactivator is preferably under the control of an inducible promoter, a tissue-specific promoter or a constitutive promoter, and a second heterologous expression cassette or parts thereof comprising a nucleotide sequence encoding a trehalose-6-phosphate phosphatase under control of a promoter capable of directing the expression of the nucleotide sequence in the plastids of said plant, e.g. a transactivator-regulated promoter wherein the corresponding transactivator is preferably under the control of an inducible promoter, a tissue-specific promoter or a constitutive promoter.
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag with instructions for use.
The invention further provides:
A heterologous plant nuclear expression cassette comprising a nucleotide sequence encoding a trehalose-6-phosphate synthase, preferably under the control of an inducible promoter, e.g., a wound inducible or chemically inducible promoter;
a vector comprising such plant expressible cassette; and
a plant transformed with such a vector.
The invention further provides:
A heterologous plant nuclear expression cassette comprising a nucleotide sequence encoding a trehalose-6-phosphate phosphatase, preferably under the control of an inducible promoter, e.g., a wound inducible or chemically inducible promoter;
a vector comprising such plant expressible cassette; and
a plant transformed with such a vector.
The invention further provides:
A heterologous plant expression cassette comprising a nucleotide sequence encoding a trehalose-6-phosphate synthase, preferably under the control of an inducible promoter, e.g., a wound inducible or chemically inducible promoter, and further comprising a nucleotide sequence encoding a trehalose-6-phosphate phosphatase, preferably under the control of an inducible promoter, e.g., a wound inducible or chemically inducible promoter;
a vector comprising such plant expressible cassette; and
a plant transformed with such a vector.
In a further embodiment, the invention encompasses expression of nucleotide sequences encoding trehalose biosynthetic enzymes in plastids under the control of a promoter capable of directing the expression of a nucleotide sequence in the plastids of a plant, e.g. a transactivator-regulated promoter, and the gene for the transactivator is in the nuclear DNA, under the control of an plant promoter. For example, plastid transformation vectors are typically constructed using a phage promoter, such as the phage T7 gene 10 promoter, the transcriptional activation of which is dependent upon an RNA polymerase such as the phage T7 RNA polymerase. The resulting line is crossed to a transgenic line containing a nuclear coding region for a phage RNA polymerase supplemented with a chloroplast-targeting sequence and operably linked to a plant promoter, preferably an inducible promoter, a tissue-specific promoter or a constitutive promoter, preferably a chemically inducible promoter such as the tobacco PR-1a promoter.
The invention thus additionally provides:
A plant comprising
a heterologous plastid expression cassette or parts thereof comprising a nucleotide sequence encoding at least one trehalose biosynthetic enzyme such as, for example, a trehalose-6-phosphate synthase or a trehalose-6-phosphate phosphatase under the control of a promoter capable of directing the expression of a nucleotide sequence in the plastids of a plant; also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
A plant comprising
a heterologous nuclear expression cassette or parts thereof preferably comprising an inducible promoter, a tissue-specific promoter or a constitutive promoter, more preferably an inducible promoter, e.g., a wound-inducible or chemically-inducible promoter, for example the tobacco PR-1a promoter, operably linked to a DNA sequence coding for a transactivator (preferably a transactivator not naturally occurring in plants, preferably a RNA polymerase or DNA binding protein, e.g., T7 RNA polymerase), said transactivator being optionally fused to a plastid targeting sequence, e.g., a chloroplast targeting sequence (e.g., a plant expressible expression cassette as described above); and
a heterologous plastid expression cassette or parts thereof comprising a transactivator-mediated promoter regulated by the transactivator (e.g., the T7 promoter when the transactivator is T7 RNA polymerase) and operably linked to a nucleotide sequence encoding at least one trehalose biosynthetic enzyme such as, for example, a trehalose-6-phosphate synthase; also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
The invention furthermore provides:
A plant comprising
a heterologous nuclear expression cassette or parts thereof preferably comprising an inducible promoter, a tissue-specific promoter or a constitutive promoter, more preferably an inducible promoter, e.g., a wound-inducible or chemically-inducible promoter, for example the tobacco PR-1a promoter, operably linked to a nucleotide sequence encoding a transactivator (preferably a transactivator not naturally occurring in plants, preferably a RNA polymerase or DNA binding protein, e.g., T7 RNA polymerase), said transactivator being optionally fused to a plastid targeting sequence, e.g., a chloroplast targeting sequence (e.g., a plant expressible expression cassette as described above); and
a heterologous plastid expression cassette or parts thereof comprising a transactivator-mediated promoter regulated by the transactivator (e.g., the T7 promoter when the transactivator is T7 RNA polymerase) and operably linked to a nucleotide sequence encoding a trehalose-6-phosphate phosphatase;
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
The invention furthermore provides:
A plant comprising
a heterologous nuclear expression cassette or parts thereof preferably comprising an inducible promoter, a tissue-specific promoter or a constitutive promoter, more preferably an inducible promoter, e.g., a wound-inducible or chemically-inducible promoter, for example the tobacco PR-1a promoter, operably linked to a DNA sequence coding for a transactivator (preferably a transactivator not naturally occurring in plants, preferably a RNA polymerase or DNA binding protein, e.g., T7 RNA polymerase), said transactivator being optionally fused to a plastid targeting sequence, e.g., a chloroplast targeting sequence (e.g., a plant expressible expression cassette as described above); and
a heterologous plastid expression cassette or parts thereof comprising a transactivator-mediated promoter regulated by the transactivator (e.g., the T7 promoter when the transactivator is T7 RNA polymerase) and operably linked to a nucleotide sequence encoding a trehalose-6-phosphate synthase and a transactivator-mediated promoter regulated by the transactivator (e.g., the T7 promoter when the transactivator is T7 RNA polymerase) and operably linked to a nucleotide sequence encoding a trehalose-6-phosphate phosphatase;
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
In a further embodiment the invention encompasses expression of nucleotide sequences encoding trehalose biosynthetic in the plastid under the control of a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase. Such promoters are e.g., but not limited to, a clpP promoter, a 16S r-RNA gene promoter, a psbA promoter or a rbcL promoter.
The invention thus additionally provides:
A plant comprising
a heterologous plastid expression cassette or parts thereof preferably comprising a promoter capable of expression of a nucleotide sequence encoding trehalose biosynthetic enzymes in plant plastids, for example a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase, operably linked to at least a nucleotide sequence encoding a trehalose biosynthetic enzyme such as, for example, a trehalose-6-phosphate synthase; also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
The invention furthermore provides:
A plant comprising
a heterologous plastid expression cassette or parts thereof preferably comprising a promoter capable of expression of a nucleotide sequence encoding a trehalose biosynthetic enzyme in plant plastids, for example a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase, operably linked to a nucleotide sequence encoding a trehalose-6-phosphate phosphatase;
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
The invention furthermore provides:
A plant comprising
a heterologous plastid expression cassette or parts thereof preferably comprising a promoter capable of expression of a nucleotide sequence encoding a trehalose biosynthetic enzyme in plant plastids, for example a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase, operably linked to a nucleotide sequence encoding a trehalose-6-phosphate synthase and a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase, operably linked to a nucleotide sequence encoding a trehalose-6-phosphate phosphatase;
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
In a further embodiment, the invention encompasses the expression from a single promoter of two or more genes in plant plastids in an operon-like polycistronic gene. In a preferred embodiment, an operon-like polycistronic gene comprises the two or more genes, e.g. genes comprising a nucleotide sequence encoding a trehalose biosynthetic enzyme, operably linked to a promoter capable of directing the expression of operon-like polycistronic gene in plastids and is inserted into the plastid genome. In a preferred embodiment, the operon-like polycistronic gene comprises an intervening DNA sequence between two genes in the operon-like polycistronic gene, preferably a DNA sequence not present in the plastid genome. In another preferred embodiment, the intervening DNA sequence is derived from the 5xe2x80x2 untranslated (UTR) region of a non-eukaryotic gene, preferably a viral 5xe2x80x2UTR, preferably a 5xe2x80x2UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage. In a preferred embodiment, the DNA sequence is modified to prevent the formation of secondary structures that inhibit or repress translation of the gene located immediately downstream of the intervening DNA sequence. In a preferred embodiment, the expression, preferably the translation, of genes located immediately downstream of the intervening DNA sequence is increased.
The invention thus furthermore provides:
A plant comprising
a heterologous nuclear expression cassette or parts thereof preferably comprising an inducible promoter, a tissue-specific promoter or a constitutive promoter, more preferably an inducible promoter, e.g., a wound-inducible or chemically-inducible promoter, for example the tobacco PR-1a promoter, operably linked to a nucleotide sequence encoding a transactivator (preferably a transactivator not naturally occurring in plants, preferably a RNA polymerase or DNA binding protein, e.g., T7 RNA polymerase), said transactivator being optionally fused to a plastid targeting sequence, e.g., a chloroplast targeting sequence (e.g., a plant expressible expression cassette as described above); and
a heterologous plastid expression cassette or parts thereof comprising a transactivator-mediated promoter regulated by the transactivator (e.g., the T7 promoter when the transactivator is T7 RNA polymerase) and operably linked to an operon-like polycistronic gene comprising at least one gene comprising a nucleotide sequence encoding a trehalose biosynthetic enzyme. In a preferred embodiment, the operon-like polycistronic gene comprises one gene comprising a nucleotide sequence encoding a trehalose phosphate synthase and one gene encoding a nucleotide sequence encoding a trehalose phosphate phosphatase. In a preferred embodiment, the operon-like polycistronic gene comprises an intervening DNA sequence between two genes in the operon-like polycistronic gene, preferably a DNA sequence not present in the plastid genome. In a preferred embodiment, the DNA sequence is derived from the 5xe2x80x2 untranslated (UTR) region of a non-eukaryotic gene, preferably a viral 5xe2x80x2UTR, preferably a 5xe2x80x2UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage. In a preferred embodiment, the DNA sequence is modified to prevent the formation of secondary structures that inhibit or repress translation of the gene located immediately downstream of the intervening DNA sequence. In a preferred embodiment, the expression, preferably the translation, of genes located immediately downstream of the intervening DNA sequence is increased.
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
The invention furthermore provides:
A plant comprising
a heterologous nuclear expression cassette or parts thereof preferably comprising a promoter capable of expression of a nucleotide sequence encoding a trehalose biosynthetic enzyme in plant plastids, for example a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase, operably linked to an operon-like polycistronic gene comprising at least one gene comprising a nucleotide sequence encoding a trehalose biosynthetic enzyme. In a preferred embodiment, the operon-like polycistronic gene comprises one gene comprising a nucleotide sequence encoding a trehalose phosphate synthase and one gene encoding a nucleotide sequence encoding a trehalose phosphate phosphatase. In a preferred embodiment, the operon-like polycistronic gene comprises an intervening DNA sequence between two genes in the operon-like polycistronic gene, preferably a DNA sequence not present in the plastid. In a preferred embodiment, the DNA sequence is derived from the 5xe2x80x2 untranslated (UTR) region of a non-eukaryotic gene, preferably a viral 5xe2x80x2UTR, preferably a 5xe2x80x2UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage. In a preferred embodiment, the DNA sequence is modified to prevent the formation of secondary structures that inhibit or repress translation of the gene located immediately downstream of the intervening DNA sequence. In a preferred embodiment, the expression, preferably the translation, of genes located immediately downstream of the intervening DNA sequence is increased.
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
The invention furthermore provides:
A plant expressible expression cassette preferably comprising an inducible promoter, e.g., a wound-inducible or chemically-inducible promoter, for example the tobacco PR-1a promoter, operably linked to a nucleotide sequence encoding a transactivator (preferably a transactivator not naturally occurring in plants, preferably a RNA polymerase or DNA binding protein, e.g., T7 RNA polymerase), said transactivator being fused to a plastid targeting sequence, e.g., a chloroplast targeting sequence;
a vector comprising such a plant expressible cassette; and
a plant transformed with such a vector or a transgenic plant the genome of which comprises such a plant expressible expression cassette.
The invention also provides:
A heterologous plastid expression cassette comprising a transactivator-mediated promoter regulated by the transactivator (e.g., the T7 promoter when the transactivator is T7 RNA polymerase) and operably linked to a nucleotide sequence encoding at least one trehalose biosynthetic enzyme such as, for example, a trehalose-6-phosphate synthase and/or a trehalose 6-phosphate phosphatase.
The invention also provides:
A heterologous plastid expression cassette comprising a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase and operably linked to a nucleotide sequence encoding at least one trehalose biosynthetic enzyme such as, for example, a trehalose-6-phosphate synthase and/or trehalose 6-phosphate phosphatase.
The invention also provides:
A heterologous plastid expression cassette comprising a promoter capable of expression of a trehalose biosynthetic gene in plant plastids, for example a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase, or a transactivator-mediated promoter regulated by the transactivator (e.g., the T7 promoter when the transactivator is T7 RNA polymerase), operably linked to an operon-like polycistronic gene comprising nucleotide sequences encoding both trehalose biosynthetic enzymes. In a preferred embodiment, the operon-like polycistronic gene comprises an intervening DNA sequence between two genes in the operon-like polycistronic gene, preferably a DNA sequence not present in the plastid genome. In a preferred embodiment, the DNA sequence is comprises a portion of the 5xe2x80x2 untranslated (UTR) region of a non-eukaryotic gene, preferably a viral 5xe2x80x2UTR, preferably a 5xe2x80x2UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage. In a preferred embodiment, the DNA sequence is modified to prevent the formation of secondary structures that inhibit or repress the translation of the gene located immediately downstream of the intervening DNA sequence. In a preferred embodiment, the expression, preferably the translation, of genes located immediately downstream of the intervening DNA sequence is increased.
The invention also comprises:
A method of producing a plant as described above comprising
pollinating a plant comprising a heterologous plastid expression cassette or parts thereof comprising a transactivator-mediated promoter regulated and operably linked to a nucleotide sequence of interest, but preferably a nucleotide sequence encoding at least one trehalose biosynthetic enzyme such as, for example a trehalose-6-phosphate synthase and/or a trehalose 6-phosphate phosphatase with pollen from a plant comprising a heterologous nuclear expression cassette or parts thereof comprising an inducible promoter, a tissue-specific promoter or a constitutive promoter, more preferably an inducible promoter, operably linked to a nucleotide sequence encoding a transactivator capable of regulating said transactivator-mediated promoter;
recovering seed from the plant thus pollinated; and
cultivating a plant as described above from said seed.
The invention further provides:
A method for producing trehalose in a plant by expressing at least one heterologous nucleotide sequence encoding a trehalose biosynthetic enzyme under the control of any one of the promoters described above, for example an inducible promoter, e.g., a wound inducible or chemically inducible promoter, in the nuclear genome of said plant or by expressing at least one heterologous nucleotide sequence encoding a trehalose biosynthetic enzyme in the plastids of said plant under the control of a promoter capable of expressing said nucleotide sequence in the plastids of said plant or in any one of the expression cassettes described above.
A method for protecting a plant against drought, high salinity, osmotic stress and temperature extremes by expressing in said plant at least one nucleotide sequence encoding a trehalose biosynthetic enzyme from the nuclear genome of said plant under the control of an inducible promoter e.g., a wound inducible or chemically inducible promoter, or from the plastid genome of said plant under the control of a promoter capable of expressing said nucleotide sequence in the plastids of said plant.
A method for increasing storage properties of harvested plants by expressing in said plant at least one nucleotide sequence encoding a trehalose biosynthetic enzyme from the nuclear genome of said plant under the control of an inducible promoter e.g., a wound inducible or chemically inducible promoter, or from the plastid genome of said plant under the control of a promoter capable of expressing said nucleotide sequence in the plastids of said plant.
A method for improving shelf-life of fruits and vegetables, and preserving flowers by expressing in said fruits, vegetables and flowers at least one nucleotide sequence encoding a trehalose biosynthetic enzyme from the nuclear genome of said plant under the control of an inducible promoter e.g., a wound inducible or chemically inducible promoter, or from the plastid genome of said plant under the control of a promoter capable of expressing said nucleotide sequence in the plastids of said plant.
A method for stabilizing proteins, preferably transgenic proteins, expressed in transgenic plants by expressing in said plant at least one nucleotide sequence encoding a trehalose biosynthetic enzyme from the nuclear genome of said plant under the control of an inducible promoter e.g., a wound inducible or chemically inducible promoter, or from the plastid genome of said plant under the control of a promoter capable of expressing said nucleotide sequence in the plastids of said plant.
The present invention further provides:
A method of expressing two or more genes from a single promoter in the plastids of a plant comprising introducing into the plastid genome of said plant a operon-like polycistronic gene comprising said two or more genes operably linked to a promoter capable of expressing said operon-like polycistronic gene in the plastids of said plant, wherein said operon-like polycistronic gene further comprises an intervening DNA sequence between two genes. In a preferred embdiment, a DNA sequence not present in the plastid genome. In a preferred embodiment, the DNA sequence is comprises a portion of the 5xe2x80x2 untranslated (UTR) region of a non-eukaryotic gene, preferably a viral 5xe2x80x2UTR, preferably a 5xe2x80x2UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage. In a preferred embodiment, the DNA sequence is modified to prevent the formation of secondary structures that inhibit or repress the translation of the gene located immediately downstream of the intervening DNA sequence. In a preferred embodiment, the expression, preferably the translation, of genes located immediately downstream of the intervening DNA sequence is increased.
In a preferred embodiment, the operon-like polycistronic gene comprises at least one gene comprising a nucleotide sequence encoding a trehalose biosynthetic gene. In another preferred embodiment, the operon-like polycistronic gene comprises a gene comprising a nucleotide sequence encoding a trehalose phosphate synthase and a gene comprising a nucleotide sequence encoding a trehalose phosphate phosphatase.
In order to ensure a clear and consistent understanding of the specification and the claims, the following definitions are provided:
xe2x80x9cDrought resistancexe2x80x9d is the trait of a transgenic trehalose-producing plant to sustain prolonged periods of time receiving less water than a wild-type (nontransgenic) plant would normally require or without being watered, and without showing the same degree of wilting of its leaves or other characteristics of dessication that appear in a wild-type plant grown under the same conditions.
xe2x80x9cGenexe2x80x9d as used herein comprises a nucleotide sequence optionally operably linked to DNA sequences preceding or following the nucleotide sequence. The nucleotide sequence is typically transcribable into RNA, such as e.g. mRNA (sense RNA or antisense RNA), rRNA, tRNA or snRNA. A nucleotide sequence in a gene optionally comprises a coding sequence, which can be translated into a polypeptide. Examples of DNA sequences preceding or following the nucleotide sequence are 5xe2x80x2 and 3xe2x80x2 untranslated sequences, termination signals and ribosome binding sites (rbs), or portions thereof. Further elements that may also be present in a gene are, for example, introns.
xe2x80x9cExpression cassettexe2x80x9d as used herein means a DNA construct designed so that a nucleotide sequence inserted herein can be transcribed and, optionally translated, in an appropriate host cell. The expression cassette typically comprises regulatory elements, such as a promoter capable of directing expression of the nucleotide sequence operably linked to the nucleotide sequence, which is itself optionally operably linked to 3xe2x80x2 sequences, such as 3xe2x80x2 regulatory sequences or termination signals. The expression cassette also may comprises sequences required for proper translation of a coding sequence comprised in the nucleotide sequence. The nucleotide sequence usually comprises the coding sequence of a protein but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA that, in the sense or antisense direction, inhibits expression of a particular gene, e.g., antisense RNA. The expression cassette comprising the nucleotide sequence of interest may be polycistronic, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue or organ or stage of development. A nuclear expression cassette is usually inserted into the nuclear genome of a plant and is capable of directing the expression of a particular nucleotide sequence from the nuclear genome of said plant. A plastid expression cassette is usually inserted in to the plastid genome of a plant and is capable of directing the expression of a particular nucleotide sequence from the plastid genome of said plant, for example a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase, or a transactivator-mediated promoter. A plastid expression cassette as described herein may optionally comprise an operon-like polycistronic gene.
xe2x80x9cRegulatory elementsxe2x80x9d refer to DNA sequences involved in the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest, and may also include 5xe2x80x2 and 3xe2x80x2 untranslated regions (UTR) or termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence, such as, in the case of expression in plastids, ribosome binding sites (rbs).
xe2x80x9cHeterologousxe2x80x9d as used herein means xe2x80x9cof different natural originxe2x80x9d or represents a non-natural state. For example, if a host cell is transformed with a nucleotide sequence derived from another organism, particularly from another species, that nucleotide sequence is heterologous with respect to that host cell and also with respect to descendants of the host cell which carry that gene. Similarly, heterologous refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. a different copy number, or under the control of different regulatory elements. A transforming nucleotide sequence may comprise a heterologous coding sequence, or heterologous regulatory elements. Alternatively, the transforming nucleotide sequence may be completely heterologous or may comprise any possible combination of heterologous and endogenous nucleic acid sequences.
xe2x80x9cExpressionxe2x80x9d refers to the transcription and/or translation of a nucleotide sequence, for example an endogenous gene or a heterolous gene, in a host organism, e.g. microbes or plants. In the case of antisense constructs, for example, expression may refer to the transcription of the antisense DNA only.
An xe2x80x9coperon-like polycistronic genexe2x80x9d comprises two or more genes of interest under control of a single promoter capable of directing the expression of such operon-like polycistronic gene in plant plastids. Every gene in a operon-like polycistronic gene optionally comprises a ribosome binding site (rbs) operably linked to the 5xe2x80x2 end of the nucleotide sequence. Preferably each rbs in the operon-like polycistronic gene is different. The operon-like polycistronic gene also typically comprises a 5xe2x80x2 UTR operably linked to the 5xe2x80x2 end of the rbs of the first gene in the operon-like polycistronic gene and a 3xe2x80x2 UTR operably linked to the 3xe2x80x2 end of the last gene in the operon-like polycistronic gene. Two genes in a operon-like polycistronic gene may also comprise several nucleic acids which overlap between the two genes.
xe2x80x9cHomoplastidicxe2x80x9d refers to a plant, plant tissue or plant cell wherein all of the plastids are genetically identical. This is the normal state in a plant when the plastids have not been transformed, mutated, or otherwise genetically altered. In different tissues or stages of development, the plastids may take different forms, e.g., chloroplasts, proplastids, etioplasts, amyloplasts, chromoplasts, and so forth.
xe2x80x9cMarker genexe2x80x9d: a gene encoding a selectable or screenable trait.
xe2x80x9cInducible Promoterxe2x80x9d: An xe2x80x9cinducible promoterxe2x80x9d is a promoter which initiates transcription only when the plant is exposed to some particular external stimulus, as distinguished from constitutive promoters or promoters specific to a specific tissue or organ or stage of development. Particularly preferred for the present invention are chemically-inducible promoters and wound-inducible promoters. Chemically inducible promoters include plant-derived promoters, such as the promoters in the systemic acquired resistance pathway, for example the PR promoters, e.g., the PR-1, PR-2, PR-3, PR4, and PR-5 promoters, especially the tobacco PR-1a promoter and the Arabidopsis PR-1 promoter, which initiate transcription when the plant is exposed to BTH and related chemicals. See U.S. Pat. No. 5,614,395, incorporated herein by reference, and WO 98/03536, incorporated herein by reference. Chemically-inducible promoters also include receptor-mediated systems, e.g., those derived from other organisms, such as steroid-dependent gene expression, copper-dependent gene expression, tetracycline-dependent gene expression, and particularly the expression system utilizing the USP receptor from Drosophila mediated by juvenile growth hormone and its agonists, described in EP-A 0 859 851, incorporated herein by reference, as well as systems utilizing combinations of receptors, e.g., as described in EP-A 0 813 604, incorporated herein by reference. Wound inducible promoters include promoters for proteinase inhibitors, e.g., the proteinase inhibitor II promoter from potato, and other plant-derived promoters involved in the wound response pathway, such as promoters for polyphenyl oxidases, LAP and TD. See generally, C. Gatz, xe2x80x9cChemical Control of Gene Expressionxe2x80x9d, Annu. Rev. Plant Physiol. Plant Mol. Biol. (1997) 48: 89-108, the contents of which are incorporated herein by reference.
xe2x80x9cOperably linked to/associated withxe2x80x9d: a DNA sequence, for example comprising a regulatory element, is said to be xe2x80x9coperably linked toxe2x80x9d or xe2x80x9cassociated withxe2x80x9d a nucleotide sequence if the two sequences are situated such that the DNA sequence affects expression of the nucleotide sequence.
xe2x80x9cPhenotypic traitxe2x80x9d: a detectable property resulting from the expression of one or more genes.
xe2x80x9cPlantxe2x80x9d: A xe2x80x9cplantxe2x80x9d refers to any plant or part of a plant at any stage of development. In some embodiments of the invention, the plants may be lethally wounded to induce expression or may be induced to express lethal levels of a desired protein, and so the term xe2x80x9cplantxe2x80x9d as used herein is specifically intended to encompass plants and plant material which have been seriously damaged or killed, as well as viable plants, cuttings, cell or tissue cultures, and seeds. Preferably, plants of the present invention are distinguished in that they are developmentally normal up to the point of induction of the trehalose biosynthetic gene.
xe2x80x9cPlant cellxe2x80x9d: a structural and physiological unit of the plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.
xe2x80x9cPlant materialxe2x80x9d: refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, pollen tubes, ovules, embryo sacs, egg cells, zygotes, embryos, seeds, plastids, mitochondria, cuttings, cell or tissue cultures, or any other part or product of a plant.
xe2x80x9cProgenyxe2x80x9d as used herein comprises all the subsequent generations obtained by self-pollination or out-crossing of a plant of the present invention.
xe2x80x9cPromoterxe2x80x9d: a DNA sequence that initiates transcription of an associated DNA sequence. The promoter region may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors.
xe2x80x9cProtoplastxe2x80x9d: isolated plant cell where the cell wall has been totally or partially removed.
xe2x80x9cRecombinant DNA moleculexe2x80x9d: a combination of DNA sequences that are joined together using recombinant DNA technology.
xe2x80x9cRecombinant DNA technologyxe2x80x9d: procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
xe2x80x9cScreenable marker genexe2x80x9d: a gene whose expression does not confer a selective advantage to a transformed cell, but whose expression makes the transformed cell phenotypically distinct from untransformed cells.
xe2x80x9cSelectable marker genexe2x80x9d: a gene whose expression in a plant cell gives the cell a selective advantage. The selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non-transformed cells. The selective advantage possessed by the transformed cells, compared to non-transformed cells, may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source. Selectable marker gene also refers to a gene or a combination of genes whose expression in a plant cell gives the cell both, a negative and a positive selective advantage.
In its broadest sense, the term xe2x80x9csubstantially similarxe2x80x9d, when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g. where only changes in amino acids not affecting the polypeptide function occur. Desirably the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%. Sequence comparisons are carried out using a Smith-Waterman sequence alignment algorithm (see e.g. Waterman, M.S. Introduction to Computational Biology: Maps, sequences and genomes. Chapman and Hall. London: 1995. ISBN 0-412-99391-0. The localS program, version 1.16, is used with following parameters: match: 1, mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. A nucleotide sequence xe2x80x9csubstantially similarxe2x80x9d to reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50xc2x0 C. with washing in 2xc3x97SSC, 0.1% SDS at 50xc2x0 C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50xc2x0 C. with washing in 1xc3x97SSC, 0.1% SDS at 50xc2x0 C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50xc2x0 C. with washing in 0.5xc3x97SSC, 0.1% SDS at 50xc2x0 C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50xc2x0 C. with washing in 0.1xc3x97SSC, 0.1% SDS at 50xc2x0 C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50xc2x0 C. with washing in 0.1xc3x97SSC, 0.1% SDS at 65xc2x0 C.
The term xe2x80x9csubstantially similarxe2x80x9d, when used herein with respect to a protein, means a protein corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, e.g. where only changes in amino acids not affecting the polypeptide function occur. When used for a protein or an amino acid sequence the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is at least 80%, more desirably 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.
xe2x80x9cTransactivatorxe2x80x9d: A xe2x80x9ctransactivatorxe2x80x9d is a protein which, by itself or in combination with one or more additional proteins, is capable of causing transcription of a coding region under control of a corresponding transactivator-mediated promoter. Examples of transactivator systems include phage T7 gene 10 promoter, the transcriptional activation of which is dependent upon a specific RNA polymerase such as the phage T7 RNA polymerase. The transactivator is typically an RNA polymerase or DNA binding protein capable of interacting with a particular promoter to initiate transcription, either by activating the promoter directly or by inactivating a repressor gene, e.g., by suppressing expression or accumulation of a repressor protein. The DNA binding protein may be a chimeric protein comprising a binding region (e.g., the GAL4 binding region) linked to an appropriate transcriptional activator domain. Some transactivator systems may have multiple transactivators, for example promoters which require not only a polymerase but also a specific subunit (sigma factor) for promotor recognition, DNA binding, or transcriptional activation. The transactivator is preferably heterologous with respect to the plant.
xe2x80x9cTransformation:xe2x80x9d Introduction of a nucleotide sequence into a cell. In particular, the stable integration of a DNA molecule into the genome of an organism of interest.
xe2x80x9cTrehalose biosynthetic enzymesxe2x80x9d are polypeptides involved in the biosynthesis of trehalose from glucose, e.g., as described herein, particularly trehalose-6-phosphate synthase which catalyses the condensation of UDP-glucose and glucose-6-phosphate into trehalose-6-phosphate or trehalose-6-phosphate phosphatase which phosphorylates trehalose-6-phosphate to trehalose. The nucleotide sequences encoding the trehalose biosynthetic enzymes are comprised in trehalose biosynthetic genes.
xe2x80x9cTrehalosexe2x80x9d is a D-glucopyranosyl-[1,1]-D-glucopyranoside. The prefered form of trehalose in the present invention is a,a-trehalose (a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside).
The present invention also encompasses cells comprising a DNA molecule of the present invention, wherein the DNA molecule is not in its natural cellular environment. In a preferred embodiment, such cells are plant cells. In another prefered embodiment, a DNA molecule of the present invention is expressible in such cells and is comprised in an expression cassette which allow their expression in such cells. In a preferred embodiment, the expression cassette is stably integrated into the DNA of such host cell. In another preferred embodiment, the expression cassette is comprised in a vector, which is capable of replication in the cell and remains in the cell as an extrachromosomal molecule.
In the present invention, trehalose biosynthetic genes from E. coli are preferably used, but trehalose biosynthetic genes from other organisms including but not limited to yeast, other lower organisms or higher organisms, such as plants, are suitable. For example, the yeast TPS1, TSL1 or TSL2 genes (U.S. Pat. No. 5,792,921), the Arabidopsis trehalose synthase gene (TPS1, accession number Y08568, Blazquez et al. Plant J (1998) 13:685-9), Arabidopsis trehalose phosphate phosphatases (Vogel et al. Plant J (1998) 13:673-83) or a Selaginella lepidophylla gene (accession number U96736).
The present invention also encompasses a plant comprising the plant cells described above. In a further embodiment, the DNA molecules of the present invention are expressible in the plant, and expression of any one of the DNA molecules of the present invention or of a functional portion or derivative thereof in transgenic plants confers production of trehalose and leads to drought tolerance, improved food quality, high levels of trehalose useful for industrial production, and other characteristics as described herein. The present invention therefore also encompasses transgenic plants which express trehalose due to the expression of any one of the DNA molecules of the present invention or of a functional portion or derivative thereof.
Plants transformed in accordance with the present invention may be monocots or dicots and include, but are not limited to, maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis thaliana, and woody plants such as coniferous and deciduous trees.
Preferred are monocot plants selected from the group consisting of maize, wheat barley, rye, sorghum, and rice. Further preferred are dicot plants selected from the group consisting of chicory, lettuce, cabbage, cauliflower, broccoli, pepper, squash, pumpkin, zucchini, melon, soybean, tomato, sugarcane, sugarbeet, sunflower, rapeseed, cotton, and alfalfa. Preferred groups of plants are plants producing vegetable, fruits and flowers.
Once a desired nucleotide sequence has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques, e.g. by recurrent selection breeding, like backcrossing. In this case, the recurrent parent in which the desired transgene is to be introgressed is first crossed to the non-recurrent parent that carries the transgene in question. The progeny of this cross is then mated back to the recurrent parent followed by selection in the resultant progeny for the transgene to be transferred from the non-recurrent parent. After three, preferably four, more preferably five or more generations of backcrosses with the recurrent parent with selection for the transgene, the progeny will be heterozygous for the transgene being transferred, but will be like the recurrent parent for most or almost all other genes.
For their expression in transgenic plants, the DNA molecules may require modification and optimization, particularly when the DNA molecules are of prokaryotic origin. It is known in the art that all organisms have specific preferences for codon usage, and the codons in the nucleotide sequence comprised in the DNA molecules of the present invention can be changed to conform with specific plant preferences, while maintaining the amino acids encoded thereby. Furthermore, high expression in plants is best achieved from coding sequences which have at least 35% GC content, and preferably more than 45%. Nucleotide sequences which have low GC contents may express poorly due to the existence of ATTTA motifs which may destabilize messages, and AATAAA motifs which may cause inappropriate polyadenylation. Although preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17: 477-498 (1989)). In addition, the nucleotide sequences are screened for the existence of illegitimate splice sites which cause message truncation. All changes required to be made within the nucleotide sequences such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described in the published patent applications EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubrizol), and WO 93/07278 (to Ciba-Geigy).
For efficient initiation of translation, sequences adjacent to the initiating methionine may require modification. For example, they can be modified by the inclusion of sequences known to be effective in plants. Joshi has suggested an appropriate consensus for plants (NAR 15: 6643-6653 (1987)) and Clontech suggests a further consensus translation initiator (1993/1994 catalog, page 210). These consensuses are suitable for use with the nucleotide sequences of this invention. The sequences are incorporated into constructions comprising the nucleotide sequence, up to and including the ATG (whilst leaving the second amino acid unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibility of modifying the second amino acid of the transgene).
In transgenic plants, the DNA molecules of the present invention, for example trehalose biosynthetic genes or genes encoding a transactivator, are driven by a promoter shown to be functional in plants. The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the DNA molecules in the desired cell.
Preferred promoters which are expressed constitutively include the CaMV 35S and 19S promoters, promoters from genes encoding actin or ubiquitin, and promoters derived from Agrobacterium, for example synthetic promoters as described in PCT/US94/12946. The DNA molecules of this invention, however, are preferably expressed under the regulation of promoters which are chemically regulated. This enables the trehalose to be synthesized only when the crop plants are treated with the inducing chemicals, thereby avoiding developmental abnormalities in the young plants. Preferred technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.
A second preferred category of inducible promoters is that which is wound inducible, permitting expression of the trehalose biosynthetic enzymes when the plant is injured, for example at harvest, or in silage or other processing. Numerous promoters have been described which are expressed at wound sites. Preferred promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), and Warner et al. Plant J. 3: 191-201 (1993).
Preferred tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. A preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth and Grula, Plant Molec. Biol. 12: 579-589 (1989)). A preferred promoter for root specific expression is that described by de Framond (FEBS 290: 103-106 (1991); EP 0 452 269 to Ciba-Geigy) and a further preferred root-specific promoter is that from the T-1 gene provided by this invention. A preferred stem specific promoter is that described in U.S. Pat. No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene.
In addition to the selection of a suitable promoter, constructions for expression of the protein in plants optionally require an appropriate transcription terminator to be attached downstream of the heterologous nucleotide sequence. Several such terminators are available and known in the art (e.g. tm1 from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of this invention. Numerous other sequences can be incorporated into expression cassettes for the DNA molecules of this invention. These include sequences which have been shown to enhance expression such as intron sequences (e.g. from Adh1 and bronze1) and viral leader sequences (e.g. from TMV, MCMV and AMV).
It may be preferable to target expression of the DNA molecules to different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be prefered. Subcellular localization of transgene encoded enzymes can be undertaken using techniques well known in the art. Typically, the DNA encoding the target peptide from a known organelle-targeted gene product is manipulated and fused upstream of the nucleotide sequence. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown. A preferred class of targeting sequences are the vacuole targeting sequences, e.g., as found on plant chitinases and proteases.
Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be preferred. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al. Biotechnology 4: 1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transfer, transformation is usually (but not necessarily) undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methatrexate) or a herbicide (basta). The choice of selectable marker is not, however, critical to the invention.
In another preferred embodiment, the DNA molecules of this invention are directly transformed into the plastid genome. Plastid transformation technology is described extensively in U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818 and 5,576,198; in PCT application nos. WO 95/16783 and WO 97/32977; and in McBride et al., Proc. Natl. Acad. Sci. USA 91: 7301-7305 (1994), all of which are incorporated herein by reference. Plastid transformation via biolistics was achieved initially in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al. (1988) Science 240: 1534-1537, incorporated herein by reference) and this approach, using selection for cis-acting antibiotic resistance loci (spectinomycin/streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes, was soon extended to Nicotiana tabacum (Svab et al. (1990) Proc. Natl. Acad. Sci. USA. 87: 8526-8530, incorporated herein by reference).
The basic technique for tobacco plastid transformation involves the particle bombardment of leaf or callus tissue or PEG-mediated uptake of plasmid DNA in protoplasts with regions of cloned plastid DNA flanking a selectable antibiotic resistance marker. The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the 156 kb tobacco plastid genome. Initially, point mutations in the plastid 16S rDNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin were utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45, incorporated herein by reference). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J. M., and Maliga, P., EMBO J. 12: 601-606 (1993), incorporated herein by reference). Substantial increases in transformation frequency were obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3xe2x80x2-adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917, incorporated herein by reference). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19, 4083-4089, incorporated herein by reference). Recently, plastid transformation of protoplasts from tobacco and the moss Physcomitrella patens has been attained using polyethylene glycol (PEG) mediated DNA uptake (O""Neill et al. (1993) Plant J. 3: 729-738; Koop et al. (1996) Planta 199: 193-201, both of which are incorporated herein by reference). Both particle bombardment and protoplast transformation are appropriate in the context of the present invention.
A DNA molecule of the present invention is inserted into a plastid expression cassette comprising a promoter capable of expressing the DNA molecule in plant plastids. A preferred promoter capable of expression in a plant plastid is a promoter isolated from the 5xe2x80x2 flanking region upstream of the coding region of a plastid gene, which may come from the same or a different species, and the native product of which is typically found in a majority of plastid types including those present in non-green tissues. Gene expression in plastids differs from nuclear gene expression and is related to gene expression in prokaryotes (described in Stern et al. (1997) Trends in Plant Sciences 2: 308-315, incorporated herein by reference). Plastid promoters generally contain the xe2x88x9235 and xe2x88x9210 elements typical of prokaryotic promoters and some plastid promoters are recognized by a E. coli-like RNA polymerase mostly encoded in the plastid genome and are called PEP (plastid-encoded RNA polymerase) promoters while other plastid promoters are recognized by a nuclear-encoded RNA polymerase (NEP promoters). Both types of plastid promoters are suitable for the present invention. Examples of plastid promoters are promoters of clpP genes, such as the tobacco clpP gene promoter (WO 97/06250, incorporated herein by reference) and the Arabidopsis clpP gene promoter (U.S. application Ser. No. 09/038,878, incorporated herein by reference). Another promoter that is capable of expressing a DNA molecule in plant plastids comes from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et al., Microbiol. Rev. 58:700-754 (1994), Shinozaki et al., EMBO J. 5:2043-2049 (1986), both of which are incorporated herein by reference). Other examples of promoters that are capable of expressing a DNA molecule in plant plastids are a psbA promoter or a rbcL promoter. A plastid expression cassette also preferably further comprises a plastid gene 3xe2x80x2 untranslated sequence (3xe2x80x2 UTR) operatively linked to a DNA molecule of the present invention. The role of untranslated sequences is preferably to direct the 3xe2x80x2 processing of the transcribed RNA rather than termination of transcription. Preferably, the 3xe2x80x2 UTR is a plastid rps16 gene 3xe2x80x2 untranslated sequence or the Arabidopsis plastid psbA gene 3xe2x80x2 untranslated sequence. In a further preferred embodiment, a plastid expression cassette comprises a poly-G tract instead of a 3xe2x80x2 untranslated sequence. A plastid expression cassette also preferably further comprises a 5xe2x80x2 untranslated sequence (5xe2x80x2 UTR) functional in plant plastids operatively linked to a DNA molecule of the present invention.
A plastid expression cassette is comprised in a plastid transformation vector, which preferably further comprises flanking regions for integration into the plastid genome by homologous recombination. The plastid transformation vector may optionally comprise at least one plastid origin of replication. The present invention also encompasses a plant plastid transformed with such a plastid transformation vector, wherein the DNA molecule is expressible in the plant plastid. The invention also encompasses a plant or plant cell, including the progeny thereof, comprising this plant plastid. In a preferred embodiment, the plant or plant cell, including the progeny thereof, is homoplasmic for transgenic plastids.
Other promoters that are capable of expressing a DNA molecule in plant plastids are transactivator-regulated promoters, preferably heterologous with respect to the plant or to the subcellular organelle or component of the plant cell in which expression is effected. In these cases, the DNA molecule encoding the transactivator is inserted into an appropriate nuclear expression cassette which is transformed into the plant nuclear DNA. The transactivator is targeted to plastids using a plastid transit peptide. The transactivator and the transactivator-driven DNA molecule are brought together either by crossing to a selected plastid-transformed line a transgenic line containing a DNA molecule encoding the transactivator supplemented with a plastid-targeting sequence and operably linked to a nuclear promoter, or by directly transforming a plastid transformation vector containing the desired DNA molecule into a transgenic line containing a DNA molecule encoding the transactivator supplemented with a plastid-targeting sequence and operably linked to a nuclear promoter. If the nuclear promoter is an inducible promoter, in particular a chemically inducible promoter, expression of the DNA molecule in the plastids of plants is activated by foliar application of a chemical inducer. Such inducible transactivator-mediated plastid expression system is preferably tightly regulatable, with no detectable expression prior to induction and exceptionally high expression and accumulation of protein following induction. A preferred transactivator is for example viral RNA polymerase. Preferred promoters of this type are promoters recognized by a single sub-unit RNA polymerase, such as the T7 gene 10 promoter, which is recognized by the bacteriophage T7 DNA-dependent RNA polymerase. The gene encoding the T7 polymerase is preferably transformed into the nuclear genome and the T7 polymerase is targeted to the plastids using a plastid transit peptide. Promoters suitable for nuclear expression of a gene, for example a gene encoding a viral RNA polymerase such as the T7 polymerase, are described infra or supra. Expression of the DNA molecules in plastids can be constitutive or can be inducible Expression of the DNA molecules in the plastids can be also organ- or tissue-specific. These different embodiment are extensively described in WO 98/11235, incorporated herein by reference. Thus, in one aspect, the present invention has coupled expression in the nuclear genome of a choroplast-targeted phage T7 RNA polymerase under control of the chemically inducible PR-1a promoter (U.S. Pat. No. 5,614,395 incorporated by reference) of tobacco to a chloroplast reporter transgene regulated by T7 gene 10 promoter/terminator sequences. For example, when plastid transformants homoplasmic for the maternally inherited trehalose biosynthetis genes are pollinated by lines expressing the T7 polymerase in the nucleus, F1 plants are obtained that carry both transgene constructs but do not express them. Synthesis of large amounts of enzymatically active protein is triggered in plastids of these plants only after foliar application of the PR-1a inducer compound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH).
In a preferred embodiment, two or more genes, e.g. trehalose biosynthetic genes, are transcribed from the plastid genome from a single promoter in an operon-like polycistronic gene. In a preferred embodiment, the operon-like polycistronic gene comprises an intervening DNA sequence between two genes in the operon-like polycistronic gene. In a preferred embodiment, the DNA sequence is not present in the plastid genome to avoid homologous recombination with plastid sequences. In another preferred embodiment, the DNA sequence is derived from the 5xe2x80x2 untranslated (UTR) region of a non-eukaryotic gene, preferably from a viral 5xe2x80x2UTR, preferably from a 5xe2x80x2UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage. In a preferred embodiment, the DNA sequence is modified to prevent the formation of RNA secondary structures in a RNA transcript of the operon-like polycistronic gene, e.g. between the DNA sequence and the rbs of the downstream gene. Such secondary structures would inhibit or repress the expression of the downstream gene, particularly the initiation of its translation. Such RNA secondary structures are predicted by determining their melting temperatures using computer models and programs such a the xe2x80x9cmfoldxe2x80x9d program version 3 (by Zuker and Turner, Washington University School of Medicine, St-Louis, Mo.) and other methods well known to one skilled in the art. Such a DNA sequence is exemplified below.
The presence of the intervening DNA sequence in the operon-like polycistronic gene increases the accessibility of the rbs of the downstream gene, thus resulting in higher rates of expression. Such strategy is applicable to any two or more genes to be transcribed from the plastid genome from a single promoter in an operon-like chimeric gene. Such genes can be part of a metabolic pathway, or are genes encoding input or output traits. Example of metabolic pathways are e.g. sugar biosynthetic pathways, such as trehalose or fructans.
In a further embodiment, the DNA molecules of the present invention are modified by incorporation of random mutations in a technique known as in-vitro recombination or DNA shuffling. This technique is described in Stemmer et al., Nature 370: 389-391 (1994) and U.S. Pat. No. 5,605,793 incorporated herein by reference. Millions of mutant copies of the nucleotide sequences are produced based on the original nucleotide sequence described herein and variants with improved properties, such as increased activity or altered specificity are recovered. The method encompasses forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide comprising the nucleotide sequence of this invention, wherein the template double-stranded polynucleotide has been cleaved into double-stranded-random fragments of a desired size, and comprises the steps of adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded template polynucleotide; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide. In a preferred embodiment, the concentration of a single species of double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the total DNA. In a further preferred embodiment, the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides. In another embodiment, the size of the double-stranded random fragments is from about 5 bp to 5 kb. In a further embodiment, the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles.
Numerous transformation vectors are available for plant transformation, and the genes of this invention can be used in conjunction with any such vectors. The selection of vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., Nucl Acids Res 18: 1062 (1990), Spencer et al. Theor Appl Genet 79: 625-631(1990)), the hpt gene which confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)).
Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below the construction of two typical vectors is described.
Construction of pCIB200 and pCIB2001: The binary vectors pCIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and was constructed in the following manner. pTJS75kan was created by NarI digestion of pTJS75 (Schmidhauser and Helinski, J Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline-resistance gene, followed by insertion of an AccI fragment from pUC4K carrying an NPTII (Messing and Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology 14: 266-276 (1990)). XhoI linkers were ligated to the EcoRV fragment of pCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptII chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and the XhoI-digested fragment was cloned into SaII-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, SstI, KpnI, BgIII, XbaI, and SaII. pCIB2001 is a derivative of pCIB200 which created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI, BgIII, XbaI, SaII, MluI, BcII, AvrII, ApaI, HpaI, and StuI. pCIB2001, in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OriT and OriV functions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
Construction of pCIB10 and Hygromycin Selection Derivatives thereof: The binary vector pCIB10 contains a gene encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-161 (1987)). Various derivatives of pCIB10 have been constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).
Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques which do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of some typical vectors is described.
Construction of pCIB3064: pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5xe2x80x2 of the start site. These sites were mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites SspI and PvuII. The new restriction sites were 96 and 37 bp away from the unique SaII site and 101 and 42 bp away from the actual start site. The resultant derivative of pCIB246 was designated pCIB3025. The GUS gene was then excised from pCIB3025 by digestion with SaII and SacI, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 was obtained from the John Innes Centre, Norwich and the a 400 bp SmaI fragment containing the bar gene from Streptomyces viridochromogenes was excised and inserted into the HpaI site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064 which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene fro ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites SphI, PstI, HindIII, and BamHI. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
Construction of pSOG19 and pSOG35: pSOG35 is a transformation vector which utilizes the E. coli gene dihydrofolate reductase (DHFR) as a selectable marker conferring resistance to methotrexate. PCR was used to amplify the 35S promoter (xcx9c800 bp), intron 6 from the maize Adh1 gene (xcx9c550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250 bp fragment encoding the E. coli dihydrofolate reductase type II gene was also amplified by PCR and these two PCR fragments were assembled with a SacI-Pstl fragment from pBI221 (Clontech) which comprised the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generated pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generated the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites available for the cloning of foreign sequences.
Gene sequences intended for expression in transgenic plants are firstly assembled in expression cassettes behind a suitable promoter and upstream of a suitable transcription terminator. These expression cassettes can then be easily transferred to the plant transformation vectors described above.
The selection of promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and this selection will reflect the desired location of biosynthesis of the enzyme. Alternatively, the selected promoter may drive expression of the gene under a light-induced or other temporally regulated promoter. A further (and preferred) alternative is that the selected promoter be inducible by an external stimulus, e.g., application of a specific chemical inducer or wounding. This would provide the possibility of inducing trehalose biosynthetic gene transcription only when desired.
A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants and include the CaMV 35S terminator, the tmI terminator, the nopaline synthase terminator, the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.
Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develep 1: 1183-1200 (1987)). In the same experimental system, the intron from the maize bronze1 gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the xe2x80x9cxcexa9-sequencexe2x80x9d), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15; 65-79 (1990)).
Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the aminoterminal end of various proteins and which is cleaved during chloroplast import yielding the mature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused to heterologous gene products to effect the import of heterologous products into the chloroplast (van den Broeck et al. Nature 313: 358-363 (1985)). DNA encoding for appropriate signal sequences can be isolated from the 5xe2x80x2 end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized.
Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting to cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).
In addition, sequences have been characterized which cause the targeting of gene products to other cell compartments. Aminoterminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler and Ho, Plant Cell 2: 769-783 (1990)). Additionally, aminoterminal sequences in conjunction with carboxyterminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the aminoterminal ATG of the transgene. The signal sequence selected should include the known cleavage site and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or alternatively replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by (Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier. pp 1081-1091 (1982); Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986)). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes. The choice of targeting which may be required for trehalose biosynthetic genes will depend on the cellular localization of the precursor required as the starting point for a given pathway. This will usually be cytosolic or chloroplastic, although it may is some cases be mitochondrial or peroxisomal.
The above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell targeting goal under the transcriptional regulation of a promoter which has an expression pattern different to that of the promoter from which the targeting signal derives.
The present invention encompasses the expression of trehalose biosynthetic genes under the regulation of any promoter that is expressible in plants, regardless of the origin of the promoter.
Furthermore, the invention encompasses the use of any plant-expressible promoter in conjunction with any further sequences required or selected for the expression of the trehalose biosynthetic gene. Such sequences include, but are not restricted to, transcriptional terminators, extraneous sequences to enhance expression (such as introns [e.g. Adh intron 1], viral sequences [e. g. TMV-xcexa9]), and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
Suitable plant-expressible promoters are those that are expressed constitutively such as the CaMV 35S promoter, the actin promoter or the ubiquitin promoter.
Construction of the plasmid pCGN1761 containing the xe2x80x9cdoublexe2x80x9d 35S promoter is described in the published patent application EP 0 392 225 (example 23). pCGN1761 contains the xe2x80x9cdoublexe2x80x9d 35S promoter and the tmI transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone. A derivative of pCGN1761 was constructed which has a modified polylinker which includes NotI and XhoI sites in addition to the existing EcoRI site. This derivative was designated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNA sequences or gene sequences (including microbial ORF sequences) within its polylinker for the purposes of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-gene sequence-tmI terminator cassette of such a construction can be excised by HindIII, SphI, SaII, and XbaI sites 5xe2x80x2 to the promoter and XbaI, BamHI and BgII sites 3xe2x80x2 to the terminator for transfer to transformation vectors such as those described above. Furthermore, the double 35S promoter fragment can be removed by 5xe2x80x2 excision with HindIII, SphI, SaII, XbaI, or PstI, and 3xe2x80x2 excision with any of the polylinker restriction sites (EcoRI, NotI or XhoI) for replacement with another promoter.
For any of the constructions described in this section, modifications around the cloning sites can be made by the introduction of sequences which may enhance translation. This is particularly useful when genes derived from microorganisms are to be introduced into plant expression cassettes as these genes may not contain sequences adjacent to their initiating methionine which may be suitable for the initiation of translation in plants. In cases where genes derived from microorganisms are to be cloned into plant expression cassettes at their ATG it may be useful to modify the site of their insertion to optimize their expression. Modification of pCGN1761ENX by optimization of the translational initiation site is described by way of example to incorporate one of several optimized sequences for plant expression (e.g. Joshi, supra).
Further plant-expressible promoters that can be suitably used within the scope of the present invention are chemically regulatable promoters such as those described hereinafter. For example, this section describes the replacement of the double 35S promoter in pCGN1761ENX with any promoter of choice; by way of example, the chemically regulatable PR-1a promoter is described in U.S. Pat. No. 5,614,395, which is hereby incorporated by reference in its entirety, and the chemically regulatable Arabidopsis PR-1 promoter is described in U.S. Provisional Application No. 60/027,228, incorporated herein by reference. The promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers which carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be resequenced to check for amplification errors after the cloning of the amplified promoter in the target vector. The chemically regulatable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (see EP 0 332 104, example 21 for construction) and transferred to plasmid pCGN1761ENX. pCIB1004 is cleaved with NcoI and the resultant 3xe2x80x2 overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with HindIII and the resultant PR-1a promoter containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is done by cleavage with XhoI and blunting with T4 polymerase, followed by cleavage with HindIII and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENX derivative with the PR-1a promoter and the tmI terminator and an intervening polylinker with unique EcoRI and NotI sites. Selected trehalose biosynthetic genes can be inserted into this vector, and the fusion products (i.e. promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described in this application.
Various chemical regulators may be employed to induce expression of the trehalose biosynthetic coding sequence in the plants transformed according to the present invention. In the context of the instant disclosure, xe2x80x9cchemical regulatorsxe2x80x9d include chemicals known to be inducers for the PR-1a promoter in plants, or close derivatives thereof. A preferred group of regulators for the chemically inducible trehalose biosynthetic genes of this invention is based on the benzo-1,2,3-thiadiazole (BTH) structure and includes, but is not limited to, the following types of compounds: benzo-1,2,3-thiadiazolecarboxylic acid, benzo-1,2,3-thiadiazolethiocarboxylic acid, cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazolecarboxylic acid amide, benzo-1,2,3-thiadiazolecarboxylic acid hydrazide, benzo-1,2,3-thiadiazole-7-carboxylic acid, benzo-1,2,3-thiadiazole-7-thiocarboxylic acid, 7-cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazole-7-carboxylic acid amide, benzo-1,2,3-thiadiazole-7-carboxylic acid hydrazide, alkyl benzo-1,2,3-thiadiazolecarboxylate in which the alkyl group contains one to six carbon atoms, methyl benzo-1,2,3-thiadiazole-7-carboxylate, n-propyl benzo-1,2,3-thiadiazole-7-carboxylate, benzyl benzo-1,2,3-thiadiazole-7-carboxylate, benzo-1,2,3-thiadiazole-7-carboxylic acid sec-butylhydrazide, and suitable derivatives thereof. Other chemical inducers may include, for example, benzoic acid, salicylic acid (SA), polyacrylic acid and substituted derivatives thereof; suitable substituents include lower alkyl, lower alkoxy, lower alkylthio, and halogen. Still another group of regulators for the chemically inducible DNA sequences of this invention is based on the pyridine carboxylic acid structure, such as the isonicotinic acid structure and preferably the haloisonicotinic acid structure. Preferred are dichloroisonicotinic acids and derivatives thereof, for example the lower alkyl esters. Suitable regulators of this class of compounds are, for example, 2,6-dichloroisonicotinic acid (INA), and the lower alkyl esters thereof, especially the methyl ester.
Constitutive Expression can also be achieved by the Actin Promoter. Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the rice Act1 gene has been cloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)). A 1.3 kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts. Furthermore, numerous expression vectors based on the Act1 promoter have been constructed specifically for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991)). These incorporate the Act1-intron 1, Adh1 5xe2x80x2 flanking sequence and Adh1-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and the Act1 intron or the Act1 5xe2x80x2 flanking sequence and the Act1 intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for the expression of trehalose biosynthetic genes and are particularly suitable for use in monocotyledonous hosts. For example, promoter containing fragments can be removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761ENX, which is then available for the insertion or specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report the rice Act1 promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).
Ubiquitin is another gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflowerxe2x80x94Binet et al. Plant Science 79: 87-94 (1991), maizexe2x80x94Christensen et al. Plant Molec. Biol. 12: 619-632 (1989)) for constitutive expression. The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol). Further, Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) which comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment. The ubiquitin promoter is suitable for the expression of trehalose biosynthetic genes in transgenic plants, especially monocotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.
Another pattern of expression for the enzymes of the instant invention is root expression. A suitable root promoter is that described by de Framond (FEBS 290: 103-106 (1991)) and also in the published patent application EP 0 452 269 (to Ciba-Geigy). This promoter is transferred to a suitable vector such as pCGN1761ENX for the insertion of a trehalose biosynthetic gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.
Wound-inducible promoters may also be suitable for the expression of trehalose biosynthetic genes. Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)) and all are suitable for use with the instant invention. Logemann et al. describe the 5xe2x80x2 upstream sequences of the dicotyledonous potato wun1 gene. Xu et al. show that a wound inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier and Lehle describe the cloning of the maize Wip1 cDNA which is wound induced and which can be used to isolated the cognate promoter using standard techniques. Similarly, Firek et al. and Warner et al. have described a wound induced gene from the monocotyledon Asparagus officinalis which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the trehalose biosynthetic genes of this invention, and used to express these genes at the sites of plant wounding.
Patent Application WO 93/07278 (to Ciba-Geigy) describes the isolation of the maize trpA gene which is preferentially expressed in pith cells. The gene sequence and promoter extend up to xe2x88x921726 from the start of transcription are presented. Using standard molecular biological techniques, this promoter or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner. In fact, fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth and Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.
Chen and Jagendorf (J. Biol. Chem. 268: 2363-2367 (1993) have described the successful use of a chloroplast transit peptide for import of a heterologous transgene. This peptide used is the transit peptide from the rbcS gene from Nicotiana plumbaginifolia (Poulsen et al. Mol. Gen. Genet. 205: 193-200 (1986)). Using the restriction enzymes DraI and SphI, or Tsp509I and SphI the DNA sequence encoding this transit peptide can be excised from plasmid prbcS-8B and manipulated for use with any of the constructions described above. The DraI-SphI fragment extends from xe2x88x9258 relative to the initiating rbcS ATG to, and including, the first amino acid (also a methionine) of the mature peptide immediately after the import cleavage site, whereas the Tsp509I-SphI fragment extends from xe2x88x928 relative to the initiating rbcS ATG to, and including, the first amino acid of the mature peptide. Thus, these fragments can be appropriately inserted into the polylinker of any chosen expression cassette generating a transcriptional fusion to the untranslated leader of the chosen promoter (e.g. 35S, PR-1a, actin, ubiquitin etc.), whilst enabling the insertion of a trehalose biosynthetic gene in correct fusion downstream of the transit peptide. Constructions of this kind are routine in the art. For example, whereas the DraI end is already blunt, the 5xe2x80x2 Tsp509l site may be rendered blunt by T4 polymerase treatment, or may alternatively be ligated to a linker or adaptor sequence to facilitate its fusion to the chosen promoter. The 3xe2x80x2 SphI site may be maintained as such, or may alternatively be ligated to adaptor of linker sequences to facilitate its insertion into the chosen vector in such a way as to make available appropriate restriction sites for the subsequent insertion of a selected trehalose biosynthetic gene. Ideally the ATG of the SphI site is maintained and comprises the first ATG of the selected trehalose biosynthetic gene. Chen and Jagendorf provide consensus sequences for ideal cleavage for chloroplast import, and in each case a methionine is preferred at the first position of the mature protein. At subsequent positions there is more variation and the amino acid may not be so critical. In any case, fusion constructions can be assessed for efficiency of import in vitro using the methods described by Bartlett et al. (In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier. pp 1081-1091 (1982)) and Wasmann et al. (Mol. Gen. Genet. 205: 446-453 (1986)). Typically the best approach may be to generate fusions using the selected trehalose biosynthetic gene with no modifications at the aminoterminus, and only to incorporate modifications when it is apparent that such fusions are not chloroplast imported at high efficiency, in which case modifications may be made in accordance with the established literature (Chen and Jagendorf; Wasman et al.; Ko and Ko, J. Biol. Chem. 267:13910-13916 (1992)).
A preferred vector is constructed by transferring the DraI-SphI transit peptide encoding fragment from prbcS-8B to the cloning vector pCGN1761ENX/Sphxe2x88x92. This plasmid is cleaved with EcoRI and the termini rendered blunt by treatment with T4 DNA polymerase. Plasmid prbcS-8B is cleaved with SphI and ligated to an annealed molecular adaptor. The resultant product is 5xe2x80x2-terminally phosphorylated by treatment with T4 kinase. Subsequent cleavage with DraI releases the transit peptide encoding fragment which is ligated into the blunt-end ex-EcoRI sites of the modified vector described above. Clones oriented with the 5xe2x80x2 end of the insert adjacent to the 3xe2x80x2 end of the 35S promoter are identified by sequencing. These clones carry a DNA fusion of the 35S leader sequence to the rbcS-BA promoter-transit peptide sequence extending from xe2x88x9258 relative to the rbcS ATG to the ATG of the mature protein, and including at that position a unique SphI site, and a newly created EcoRI site, as well as the existing NotI and XhoI sites of pCGN1761ENX. This new vector is designated pCGN1761/CT. DNA sequences are transferred to pCGN1761/CT in frame by amplification using PCR techniques and incorporation of an SphI, NSphI, or NlaIII site at the amplified ATG, which following restriction enzyme cleavage with the appropriate enzyme is ligated into SphI-cleaved pCGN1761/CT. To facilitate construction, it may be required to change the second amino acid of cloned gene, however, in almost all cases the use of PCR together with standard site directed mutagenesis will enable the construction of any desired sequence around the cleavage site and first methionine of the mature protein.
A further preferred vector is constructed by replacing the double 35S promoter of pCGN 1761 ENX with the BamHI-SphI fragment of prbcS-8A which contains the full-length light regulated rbcS-8A promoter from xe2x88x921038 (relative to the transcriptional start site) up to the first methionine of the mature protein. The modified pCGN1761 with the destroyed SphI site is cleaved with Pst1 and EcoRI and treated with T4 DNA polymerase to render termini blunt. prbcS-8A is cleaved SphI and ligated to the annealed molecular adaptor of the sequence described above. The resultant product is-5xe2x80x2-terminally phosphorylated by treatment with T4 kinase. Subsequent cleavage with BamHI releases the promoter-transit peptide containing fragment which is treated with T4 DNA polymerase to render the BamHI terminus blunt. The promoter-transit peptide fragment thus generated is cloned into the prepared pCGN1761ENX vector, generating a construction comprising the rbcS-8A promoter and transit peptide with an SphI site located at the cleavage site for insertion of heterologous genes. Further, downstream of the SphI site there are EcoRI (re-created), NotI, and XhoI cloning sites. This construction is designated pCGN1761 rbcS/CT.
Similar manipulations can be undertaken to utilize other GS2 chloroplast transit peptide encoding sequences from other sources (monocotyledonous and dicotyledonous) and from other genes. In addition, similar procedures can be followed to achieve targeting to other subcellular compartments such as mitochondria.
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques which do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al, EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. The many crop species which are routinely transformable by Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean, alfalfa and poplar (EP 0 317 511 (cotton [1313]), EP 0 249 432 (tomato, to Calgene), WO 87/07299 (Brassica, to Calgene), U.S. Pat. No. 4,795,855 (poplar)). Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hxc3x6fgen and Willmitzer, Nucl. Acids Res. 16: 9877(1988)).
Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complex vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
Patent Applications EP 0 292 435 ([1280/1281] to Ciba-Geigy), EP 0 392 225 (to Ciba-Geigy) and WO 93/07278 (to Ciba-Geigy) describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, application WO 93/07278 (to Ciba-Geigy) and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for the transformation of xc3xa9lite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al., Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).
Patent Application EP 0 332 581 (to Ciba-Geigy) describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation was been described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga and Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont Biolistics(copyright) helium device using a burst pressure of xcx9c1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS+1 mg/liter NM, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as xe2x80x9cGA7sxe2x80x9d which contained half-strength MS, 2% sucrose, and the same concentration of selection agent. Patent application 08/147,161 describes methods for wheat transformation and is hereby incorporated by reference.
The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.